Fig 1: Neurogenic hypertension and cardiac mitochondrial defective OXPHOS subunit assembly/import. (A) Left, Western blot showed mitochondrial OXPHOS complex protein level in control and NG-HTN whole hearts lysate (total OXPHOS WB antibody cocktail, cat# ab110413, Abcam). Right, (i–iii) WB quantification of mitochondrial Complex-I/III, Complex-IV/III, and Complex-V/III ratio, respectively. (B) Left, Western blots showed mitochondrial OXPHOS complex protein level in cytosol vs. mitochondria fraction in control and NG-HTN hearts. Right, (i–iii) Western blot quantification of mitochondrial Complex-I/III, Complex-IV/III, and Complex-V/III ratio, respectively. Values are mean ± SEM, dot represents n = 6 rats, *P < 0.05 vs. Control, (A), Student’s t-test, (B); two-way ANOVA with Tukey’s multiple comparison test.
Fig 2: Relative levels of mtDNA and a subset of oxidative phosphorylation (OXPHOS) subunits in rat heart mitochondria from four experimental groups. (A). Relative amount of the mtDNA copy number in the heart tissue of four animal groups. The relative copy number of each group was calculated as the ratio between the mtDNA and nuclear DNA copy numbers. Data are expressed as mean values ± standard error from six independent experiments. (B). Representative Western blot of typical subunits of complexes I–V of the respiratory chain (CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTCO1, and CV-ATP5A) in the mitochondria (10 μg of protein per lane) isolated from the hearts of experimental rats. The analysis was carried out using the Total OXPHOS Rodent WB Antibody Cocktail (Abcam, #ab110413, Cambridge, UK) and polyclonal anti-VDAC1 antibodies (Abcam, #ab15895, Cambridge, UK). (C). Densitometric analysis of the content of the OXPHOS subunits in the heart mitochondria of rats from four experimental groups. Data are expressed as mean values ± standard deviations of three independent experiments. * p < 0.05 compared to the control group (CTR). Control values are taken as 100%.
Fig 3: Western blot mitochondrial complex protein expression in mitochondria and homogenates isolated from fresh or frozen cortex tissues: As illustrated in Figure 3, the male and female mice were subjected to sham or CCI injury (N = 4-6; Equal male and females). The ipsilateral injured cortex punch (4mm) was split into two equal parts. One fraction was snap frozen and stored at −80⁰C and the remaining one was used for fresh mitochondria and homogenate preparation. The extracts were lysed in RIPA buffer and were analyzed for protein expression using Western Blot analysis using an Oxphos antibody cocktail (Abcam #ab110413). All 5 complexes in mitochondria (FSHT-Mito) from fresh tissues showed a significant reduction in CCI as compared to sham animals (A Left panel). Whereas the mitochondria (FRZT-Mito) prepared from frozen cortices (A right panel) after 4 days didn’t show any significant differences between the sham and CCI group of animals. Similarly, homogenates (FSHT-Homog) (C Left panel) prepared from fresh tissues showed a significant decrease in complexes I, II, III, and IV expression but there were no changes in the (FRZT-Homog) homogenates prepared from the frozen tissues (C right panel). (B and D show the quantitative analysis of figures A and C respectively. Protein expression data are presented as N = 4-6 ± SD. Data were analyzed by one-way ANOVA with Fisher’s LSD test. * p,0.05, ** p < 0.01, *** p < 0.001.
Fig 4: Hepatic miR-33 deficiency improves mitochondrial function and homeostasis.(A) Western blot and densitometric analysis of different mitochondrial subunits blotted with the Total OXPHOS Rodent WB Antibody Cocktail (Abcam ab110413) and housekeeping standard VINCULIN in WT and HKO livers from mice fed with CD-HFD for 6 months (n = 6). (B) qPCR analysis of mitochondrial DNA and nuclear DNA in WT and HKO livers. Data represented as mtDNA/nDNA (n = 6). (C) Activity of the ETC complex I and complex II in MASH livers. Enzyme activities are expressed as change in absorbance/min/μg protein/citrate synthase activity (n = 4–6). (D) Representative electron micrographs of mitochondria profiles in WT and HKO hepatocytes from MASH livers. (E–H) Mitochondrial coverage (E), mitochondrial density (F), cumulative distribution and mean of mitochondrial area (G), and mitochondria aspect ratio (H) from WT and HKO hepatocytes (n = 3–4). (I) Western blot of PGC1α, TFAM, MFN2, OPA1, and housekeeping standard VINCULIN or GAPDH in WT and HKO livers. Data represent the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons and 2-way ANOVA followed by multiple comparison (B, C, and I).
Fig 5: Content of the subunits of five respiratory chain complexes (CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTCO1, and CV-vATP5A) in rat heart mitochondria from the experimental groups. (A) Representative Western blot of typical oxidative phosphorylation (OXPHOS) subunits of complexes I–V in the mitochondria isolated from the organs of experimental animals. The analysis was performed using the Total OXPHOS Rodent WB Antibody Cocktail and anti-VDAC1 antibodies (Abcam, #ab110413 and #ab15895, Cambridge, UK; 10 μg of mitochondrial protein per lane). PC–positive control (Abcam, #ab110341, Cambridge, UK). (B) Densitometric analysis of the level of the OXPHOS subunits in the cardiac mitochondria of experimental animals. Data are expressed as mean values ± standard errors. Control values are taken as 100%. Significant differences tested with a one-way analysis of variance and Tukey’s post hoc test. * p < 0.05.
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